Strong promoters are desired for high level recombinant protein production, in order to provide large quantities of a desired recombinant protein permitting a wide range of possible uses including industrial processes, diagnosis and disease treatment.
A typical promoter used for recombinant protein production contains a promoter element, an immediate upstream enhancer and if required other cis-acting regulatory elements. Optionally, transcription factors, which work in synergy to enhance transcription activity, are recruited to the site by specific sequence motifs.
Increasing recombinant protein expression through improvements in transcription and through preventing promoter silencing is desirable for optimizing yield. In order to be able to provide functional proteins, mammalian proteins are often expressed in mammalian cell lines as these can ensure the required post-translational modifications, such as “native” mammalian glycosylation patterns and molecular folding steps. Thus, mammalian cells are an important host for the production of clinically relevant recombinant proteins. The most widely used approach for this purpose is to establish a cell line with an actively expressed recombinant gene stably integrated in its genome. Alternatively, proteins can be transiently produced in cells for a limited period of time without the necessity of recombinant gene integration.
Chinese hamster ovary cells (CHO) cells have been the most commonly used mammalian host for large-scale commercial production of therapeutic proteins. The first CHO cell line was derived through single cell cloning in 1957. The cell line CHO-K1 was later derived from this ancestral cell line and it contains a slightly lower amount of DNA than the original CHO. Subsequently, another derivative of the original CHO cell line was mutagenized to yield CHO-DG44, a cell line with deletions of both dhfr alleles. While CHO cells are used in studies of genetics, toxicity screening, nutrition and gene expression, the most prominent use is recombinant protein expression.
Process development using CHO cell lines focuses on achieving the maximum amount of active product. Optimization of the amount of active product can be achieved via increasing the specific productivity (i.e., the product per cell) and/or by cell line development. Cell line development may include both sub-cloning the cell line to select higher producing clones and use of gene amplification.
Another way to achieve higher recombinant protein yields is to increase the cell yield (i.e., cells per volume) of the process. This may be accomplished through process development (e.g., batch, fed-batch, perfusion, etc.) and medium development. By increasing the cells per volume per day, higher levels of product may be produced.
However, even though efficiency and output in recombinant protein production has substantially increased in recent years, there is still the need in the art for alternative methods that allow an even higher expression level.